It is now possible to direct the differentiation of pluripotent human stem cells into cardiomyocytes at a scale suitable for large experiments or small clinical trials. Despite this success, there are large gaps in our understanding of how signaling pathways converge on the nucleus to control cardiac differentiation. Furthermore, stem cell-derived cardiomyocytes are currently at a fetal stage of maturity, which may be poorly suited for modeling adult diseases or tissue repair. Project 3 addresses these issues with the following aims. In Aim 1, we explore the biology of three candidate novel regulators of heart development identified by ChlPseq experiments. These include two transcription factors (MEIS2 and H0XB2) and a novel Wnt/b-catenin regulating protein (TMEM88). The genes will be knocked down in differentiating hESCs and in developing zebrafish embryos to determine their roles in cardiac differentiation and morphogenesis. In Aim 2 we will utilize two lines of genetically modified hESCs that activate fluorescent protein expression when they differentiate to mesoderm (BRACHYURY knock-in) or first heart field cells (TBX5 knock-in). We will determine how their fates become restricted during differentiation in 2D and 3D culture and after transplantation in vivo. Additionally, we will use a Wnt/b-catenin reporter to track how this pathway positively and negatively regulates formation of these key cell types. Aim 3 addresses the issue of cardiomyocyte maturation, beginning by exploring the role of 4 candidate miRNAs that are up-regulated during maturation. Next we will identify novel maturation-associated miRNAs through RNA-seq, and we will assess their function using over-expression, depletion and novel miRNA sensors.